Cell Culturing
Cells generally exist as three-dimensional aggregates in the body, but in classical plate culturing, cells are cultured in a monolayer fashion with the cells attached to a vessel. A variety of culturing methods using culturing plates and the like have been developed in the past for culturing of adherent cells. When such plate culturing methods are carried out, the cultured cells continue their growth to a “confluent” state beyond which they are unable to grow, whereupon growth halts. A problem that occurs with many cells, though it depends on the type of cell, is that when the confluent state is continued without subculturing, spontaneous detachment of the cells begins after a certain period of time, making subculturing of the cells impossible.
With growing interest in cell grafting for regenerative medicine and cellular production of substances, there is an increasing demand for methods of culturing adherent cells. In the past, various three-dimensional culturing and support culturing systems have been developed, including pseudosuspension culturing with supports such as microcarriers, spheroid culturing using modified surfaces, and hollow fiber culturing using hollow fibers as cell culture spaces. Hollow fiber culturing has been adopted as a methodology for steady-state and long term culturing of cells in an environment protected by strong hollow fibers, and many attempts are being made to achieve longer-term culturing by microcarrier culturing as well, using air lift methods or device modifications designed to accomplish continuous culturing.
Such methodologies are intended to enlarge the space in which proliferation occurs by providing a three-dimensional environment in the growth space created by a support, in order to protect the support itself by a robust environment using device modifications or methodological modifications in the large growth space, and to allow prolonging of the culturing period. The systems that employ such methods require complex apparatuses or large-volume apparatuses, and in many cases they are closed systems in which it is difficult to add to the culturing environment itself once culturing has begun. Consequently, while a methodology is desired that would allow long term culturing of cells and convenient handling of the system, no suitable methodology has yet been developed.
On the other hand, investigation of the use of three-dimensional supports for long term cell culturing methods that mimic in vivo organs has been reported, as in NPLs 1 and 2. Such methods include experiments wherein reconstructed pancreatic islets of Langerhans are embedded in vivo, as well as long-term in vitro culturing of bone marrow cells, as examples of research aimed at site-selective reconstruction. These are important achievements that have demonstrated the importance and value of cell culturing in a three-dimensional environment for long term culturing, but since such supports are highly specific for a given purpose and the materials used have a fibrous structure composed of biocompatible materials, or a higher-order structure constructed by plotting, they have poor flexibility of use and also a lack of general handleability. It is desirable to develop a methodology that is convenient and can be applied to a variety of situations.
Cells are largely classified into two types, suspended cells and adherent cells, based on the features of their living form. Both types of cells, when provided for artificial culturing, are subjected to a cycle of cell seeding, culturing, proliferation, subculturing and frozen storage.
In recent years, with increasing interest being directed toward cultured cell-based production of vaccines and in vivo proteins such as enzymes, hormones, antibodies and cytokines, as well as cell grafts for use in regenerative medicine, advances continue to be made in the development of efficient and convenient methods for mass cell culturing. Cell culturing methods using supports have received special attention from the viewpoint of efficiency, as well as attractiveness and general utility, and a wide variety of methods continue to be developed. The general culturing of these cells is also being studied from multiple standpoints, also as relates to the techniques for cryopreservation of the cells. Beyond the classical freezing methods, there have been reported techniques that attempt to achieve freezing of cells more conveniently on the culture plate itself (PTL 4), methods aimed at improving survival rates of cells in freezing procedures by using three-dimensional supports (PTL 5), examples in which fibrous supports have been used to improve the cryopreservation properties of cultured cells (PTL 6), and examples of verifying the survival efficiency of stem cells (NPL 3).
The cryopreservation media, however, have been limited to fibrous materials with special structures, and such materials, while functioning as cryopreservation media, cannot be directly used as cell culture supports and have only been utilized for temporary preservation. There is a demand for establishment of a new methodology that allows thawing of cells from cryopreservation (cell arousal) to be carried out conveniently and efficiently, and in a consistent manner from use to re-freezing.
Porous Polyimide Film
The term “polyimide” is a general term for polymers including imide bonds in the repeating unit. An “aromatic polyimide” is a polymer in which aromatic compounds are directly linked by imide bonds. An aromatic polyimide has an aromatic-aromatic conjugated structure via an imide bond, and therefore has a strong rigid molecular structure, and since the imide bonds provide powerful intermolecular force, it has very high levels of thermal, mechanical and chemical properties.
Porous polyimide films have been utilized in the prior art for filters and low permittivity films, and especially for battery-related purposes, such as fuel cell electrolyte membranes and the like. PTLs 7 to 9 describe porous polyimide films with numerous macro-voids, having excellent permeability for gases and the like, high porosity, excellent smoothness on both surfaces, relatively high strength and, despite high porosity, also excellent resistance against compression stress in the film thickness direction.